Cell culturing methods are grouped into 1) monolayer culture, 2) suspension culturing and 3) embed culturing, depending on the carrier employed. In the monolayer culturing, the cells adhere, extend and proliferate on a culture plate made of glass, plastics or the like. In general, cells can proliferate only when they adhere to a carrier, so that the monolayer culturing is the most widely employed culturing method at present. On the other hand, in the case of culturing cells which do not necessitate the adhesion to a carrier, suspension culturing is generally employed wherein cells are cultured in suspended state in a culture medium. From the viewpoint of the efficiency of the culture, suspension culturing is most suitable. For adhesion-dependent cells, microcarrier culturing has been proposed, which is inbetween the monolayer culturing and the suspension culturing. However, it has been found that some kinds of cells, particularly most of the epithelial cells, cannot express their differentiation and proliferation functions in the monolayer culturing and the microcarrier culturing. Thus, a culturing method by which the differentiation and proliferation functions can be expressed is intensively studied.
It is considered that the cells normally differentiate and proliferate in the body because the microenvironment of the cells are suitable for the cells. More particularly, the facts that 1) the capillary system is streched around the cells and the feeding of nutrients to, and removal of wastes from the cells are efficiently effected in vivo, 2) different kinds of cells coexist and they interact with each other in vivo, and 3) cells exist in the mesenchyme consisting mainly of collagen fibers in vivo, so that they exist in a three-dimensional state, efficiently control the differentiation and proliferation of the cells. Application of the enviroment similar to that in the body to a culturing system is studied. For example, as a simulation of the capillary system controlling the feeding of nutrients and removal of wastes in vivo, a culturing system in which cells are cultured on the membranes of hollow fibers, and in which the feeding of nutrients and the removal of the wastes are conducted through the membranes of the hollow fibers, has been proposed and culturing with higher density than the conventional monolayer culturing has been accomplished. However, this culturing which employs the hollow fibers is a kind of the monolayer culturing. On the other hand, as a simulation of the mesenchyme, three-dimensional cell culture (embedded culturing) in collagen sponge or in collagen gel is intensively studiedrecently. By employing the collagen gel culturing method, differentiation and proliferation of the corpus mammae epithelium cells, hepatic parenchyma cells, salivary gland cells, thymus cells and pancreas cells, which have difficulties in expression of their differentiation and proliferation functions by the conventional monolayer culturing were first accomplished. In the conventional monolayer culturing, because the cells are cultured on the surface of the substrate, the cells have flat shapes which are utterly different from the normal morphology of the cells in vivo. In contrast, in the collagen gel culturing method, the morphology of the cells resembles that of the cells in vivo. On the other hand, while the cell proliferation is stopped upon complete covering of the surface by the cells in the monolayer culturing wherein the cells proliferate on the surface of the substrate, in the collagen gel culturing (three-dimensional culturing), the cells can consistently grow without changing the morphology. Thus, the embedded culturing represented by the collagen gel culturing provides an environment which is similar to the microenvironment in vivo. As a result, the environment suitable for expression of the differentiation and proliferation functions of the cells seems to be provided.
The above-mentioned facts indicate that the two elements, i.e., formation of the nutrients-feeding and wastes-removing route and the formation of the three-dimensional cell culturing carrier, are very important for the cell differentiation.
Although the differentiation and the proliferation of the cells which were difficult in the conventional monolayer culturing have been successfully attained by the three-dimensional culturing using the collagen gel, this culturing method has problems in that the mechanical strength of the collagen gel is small and the collagen gel is difficult to shape into various forms. These are big problems when, for examples, the culturing method is applied to a large scale culturing.
The object of the present invention is to provide a carrier for cell culture which satisfies the above-mentioned two elements and which also overcomes the above-mentioned problems in the collagen gel culturing.